Pancreatic Cancer Cell Lines Research Articles

PITX2 functions as a transcription factor for GPX4 and protects pancreatic cancer cells from ferroptosis

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer.

Phenotypic, Genomic, and Transcriptomic Heterogeneity in a Pancreatic Cancer Cell Line.

To evaluate the suitability of the MIA PaCa-2 cell line for studying pancreatic cancer intratumor heterogeneity, we aim to further characterize the nature of MIA PaCa-2 cells' phenotypic, genomic, and transcriptomic heterogeneity. MIA PaCa-2 single-cell clones were established through flow cytometry. For the phenotypic study, we quantified the cellular morphology, proliferation rate, migration potential, and drug sensitivity of the clones. The chromosome copy number and transcriptomic profiles were quantified using SNPa and RNA-seq, respectively. Four MIA PaCa-2 clones showed distinctive phenotypes, with differences in cellular morphology, proliferation rate, migration potential, and drug sensitivity. We also observed a degree of genomic variations between these clones in form of chromosome copy number alterations and single nucleotide variations, suggesting the genomic heterogeneity of the population, and the intrinsic genomic instability of MIA PaCa-2 cells. Lastly, transcriptomic analysis of the clones also revealed gene expression profile differences between the clones, including the uniquely regulated ITGAV , which dictates the morphology of MIA PaCa-2 clones. MIA PaCa-2 is comprised of cells with distinctive phenotypes, heterogeneous genomes, and differential transcriptomic profiles, suggesting its suitability as a model to study the underlying mechanisms behind pancreatic cancer heterogeneity.

The scRNA-sequencing landscape of pancreatic ductal adenocarcinoma revealed distinct cell populations associated with tumor initiation and progression

Pancreatic ductal adenocarcinoma (PDAC) stands as a formidable malignancy characterized by its profound lethality. The comprehensive analysis of the transcriptional landscape holds immense significance in understanding PDAC development and exploring novel treatment strategies. However, due to the firm consistency of pancreatic cancer samples, the dissociation of single cells and subsequent sequencing can be challenging. Here, we performed single-cell RNA sequencing (scRNA-seq) on 8 PDAC patients with different lymph node metastasis status. We first identified the crucial role of MMP1 in the transition from normal pancreatic cells to cancer cells. The knockdown of MMP1 in pancreatic cancer cell lines decreased the expression of ductal markers such as SOX9 while the overexpression of MMP1 in hTERT-HPNE increased the expression of ductal markers, suggesting its function of maintaining ductal identity. Secondly, we found a S100A2+ tumor subset which fueled lymph node metastasis in PDAC. The knockdown of S100A2 significantly reduced the motility of pancreatic cancer cell lines in both wound healing and transwell migration assays. While overexpression of S100A2 led to increased migratory capability. Moreover, overexpression of S100A2 in KPC1199, a mouse pancreatic cancer cell line, caused a larger tumor burden in a hemi-spleen injection model of liver metastasis. In addition, epithelial-mesenchymal transition-related genes were decreased by S100A2 knockdown revealed by bulk RNA sequencing. We also identified several pivotal contributors to the pro-tumor microenvironment, notably OMD+ fibroblast and CCL2+ macrophage. As a result, our study provides valuable insights for early detection of PDAC and promising therapeutic targets for combatting lymph node metastasis.

Human implantable tertiary lymphoid organs (TLO) for solid tumor therapy: from organ chips to the clinic?

Abstract We previously created an in vitro human TLO model from patient-derived, circulating immune cells using organ-on-a-chip devices and 3D culture in extracellular matrix (ECM). B cells in these TLOs express activation induced cytidine deaminase, which is only expressed in lymphoid tissues and is required for class switching and somatic hypermutation (SHM). When challenged by vaccines, these TLO undergo SHM and produce antigen-specific antibodies and CD8 T cells. To model cancer associated TLOs, we integrated human pancreatic and lung cancer cell lines into these TLO Chips to understand how the immune context of hot and cold tumors differentially impacts TLO formation. The high PDL1/L2 expressing lung cancer cell line that displayed a “hot” phenotype in published murine studies induced high levels of cytokines and stimulated increased TLO formation in the lymphoid Organ Chip. In contrast, the cold, low PDL1/L2 expressing lung and pancreatic cell lines did not induce this cytokine signature or TLO formation. Importantly, TLO assembly correlated with increased B cell activation, anti-tumor CD8 activity, and tumor cell death. When the same mix of immune cells and ECM was injected in humanized mice bearing subcutaneous tumors from the hot lung cancer cell line, new lymphoid tissue containing dense aggregates of T and B cells was induced, showing for the first time that direct injection of synthetic TLOs at the tumor site may offer an alternative form of therapy for solid tumors.

Calpain 8 as a potential biomarker regulates the progression of pancreatic cancer via EMT and AKT/ERK pathway

Calpain is a non-lysozyme, calcium-dependent intracellular cysteine protease that has been shown to play a role in tumor proliferation, survival, migration, invasion, and apoptosis. Dysregulation of calpain expression is closely related to tumorigenesis. However, the role of calpain-8 (CAPN8), as a member of the calpain family, in pancreatic cancer (PC) is remains unclear. In elucidating the mechanism of CAPN8 in PC, a comprehensive bioinformatics analysis and in vitro experiments were conducted. The TCGA database was used to explore the expression level of CAPN8, and the results in PC tissues and cell lines were verified. Then, the correlation between CAPN8 and clinicopathological features was analyzed. Additionaly, promoter methylation, immune infiltration, and GO/KEGG enrichment analyses were performed. Lastly, the molecular mechanism of CAPN8 in PC was investigated by using cell counting kit (CCK) 8, transwell, wound healing, Western blot assays, and so on. Results indicate that CAPN8 was highly expressed in PC and correlated with poor prognosis and advanced TNM stage. In addition, a low level of immune infiltration was closely associated with the high expression level of CAPN8. Based on these findings, we hypothesized that CAPN8 is a potential biomarker that regulates progression of PC via EMT and the AKT/ERK pathway. SignificanceThrough comprehensive biological information and in vitro experiments, CAPN8 has been confirmed to play an important role in regulating pancreatic cancer (PC) proliferation, migration and invasion. CAPN8 is found to be closely related to the diagnosis, survival and prognosis of PC. Above all, CAPN8 may be a potential biomarker for the diagnosis and prognosis of PC.

Exosomal HSPB1, interacting with FUS protein, suppresses hypoxia-induced ferroptosis in pancreatic cancer by stabilizing Nrf2 mRNA and repressing P450.

Ferroptosis is a new type of programmed cell death, which has been involved in the progression of tumours. However, the regulatory network of ferroptosis in pancreatic cancer is still largely unknown. Here, using datasets from GEO and TCGA, we screened HSPB1, related to the P450 monooxygenase signalling, a fuel of ferroptosis, to be a candidate gene for regulating pancreatic cancer cell ferroptosis. We found that HSPB1 was enriched in the exosomes derived from human pancreatic cancer cell lines SW1990 and Panc-1. Then, hypoxic SW1990 cells were incubated with exosomes alone or together with HSPB1 siRNA (si-HSPB1), and we observed that exosomes promoted cell proliferation and invasion and suppressed ferroptosis, which was reversed by si-HSPB1. Moreover, we found a potential binding affinity between HSPB1 and FUS, verified their protein interaction by using dual-colour fluorescence colocalization and co-IP assays, and demonstrated the promoting effect of FUS on oxidative stress and ferroptosis in hypoxic SW1990 cells. Subsequently, FUS was demonstrated to bind with and stabilize the mRNA of Nrf2, a famous anti-ferroptosis gene that negatively regulates the level of P450. Furthermore, overexpressing FUS and activating the Nrf2/HO-1 pathway (using NK-252) both reversed the inhibitory effect of si-HSPB1 on exosome functions. Finally, our invivo studies showed that exosome administration promote tumour growth in nude mice of xenotransplantation, which was able to be eliminated by knockdown of HSPB1. In conclusion, exosomal HSPB1 interacts with the RNA binding protein FUS and decreases FUS-mediated stability of Nrf2 mRNA, thus suppressing hypoxia-induced ferroptosis in pancreatic cancer.

A novel multidrug-resistant cell line from a Chinese patient with pancreatic ductal adenocarcinoma

Chemotherapy resistance poses clinical challenges in pancreatic cancer treatment. Developing cell lines resistant to chemotherapy is crucial for investigating drug resistance mechanisms and identifying alternative treatment pathways. The genetic and biological attributes of pancreatic cancer depend on its aetiology, racial demographics and anatomical origin, underscoring the need for models that comprehensively represent these characteristics. Here, we introduce PDAC-X2, a pancreatic cancer cell line derived from Chinese patients. We conducted a comprehensive analysis encompassing the immune phenotype, biology, genetics, molecular characteristics and tumorigenicity of the cell line. PDAC-X2 cells displayed epithelial morphology and expressed cell markers (CK7 and CK19) alongside other markers (E-cadherin, Vimentin, Ki-67, CEA and CA19-9). The population doubling time averaged around 69 h. In vivo, PDAC-X2 cells consistently maintained their tumorigenicity, achieving a 100% tumour formation rate. Characterised by a predominantly tetraploid karyotype, this cell line exhibited a complex genetic markup. Notably, PDAC-X2 cells demonstrated resistance to multiple drugs, including gemcitabine, paclitaxel, 5-fluorouracil and oxaliplatin. In conclusion, PDAC-X2 presents an invaluable preclinical model. Its utility lies in facilitating the study of drug resistance mechanisms and the exploration of alternative therapeutic approaches aimed at enhancing the prognosis of this tumour type.

Evaluation of Helium Ion Radiotherapy in Combination with Gemcitabine in Pancreatic Cancer In Vitro.

Pancreatic cancer is one of the most aggressive and lethal cancers. New treatment strategies are highly warranted. Particle radiotherapy could offer a way to overcome the radioresistant nature of pancreatic cancer because of its biological and physical characteristics. Within particles, helium ions represent an attractive therapy option to achieve the highest possible conformity while at the same time protecting the surrounding normal tissue. The aim of this study was to evaluate the cytotoxic efficacy of helium ion irradiation in pancreatic cancer in vitro. Human pancreatic cancer cell lines AsPC-1, BxPC-3 and Panc-1 were irradiated with photons and helium ions at various doses and treated with gemcitabine. Photon irradiation was performed with a biological cabin X-ray irradiator, and helium ion irradiation was performed with a spread-out Bragg peak using the raster scanning technique at the Heidelberg Ion Beam Therapy Center (HIT). The cytotoxic effect on pancreatic cancer cells was measured with clonogenic survival. The survival curves were compared to the predicted curves that were calculated via the modified microdosimetric kinetic model (mMKM). The experimental relative biological effectiveness (RBE) of helium ion irradiation ranged from 1.0 to 1.7. The predicted survival curves obtained via mMKM calculations matched the experimental survival curves. Mainly additive cytotoxic effects were observed for the cell lines AsPC-1, BxPC-3 and Panc-1. Our results demonstrate the cytotoxic efficacy of helium ion radiotherapy in pancreatic cancer in vitro as well as the capability of mMKM calculation and its value for biological plan optimization in helium ion therapy for pancreatic cancer. A combined treatment of helium irradiation and chemotherapy with gemcitabine leads to mainly additive cytotoxic effects in pancreatic cancer cell lines. The data generated in this study may serve as the radiobiological basis for future experimental and clinical works using helium ion radiotherapy in pancreatic cancer treatment.

Calix[6]arene dismantles extracellular vesicle biogenesis and metalloproteinases that support pancreatic cancer hallmarks

Many challenges are faced in pancreatic cancer treatment due to late diagnosis and poor prognosis because of high recurrence and metastasis. Extracellular vesicles (EVs) and matrix metalloproteinases (MMPs), besides acting in intercellular communication, are key players in the cancer cell plasticity responsible for initiating metastasis. Therefore, these entities provide valuable targets for the development of better treatments. In this context, this study aimed to evaluate the potential of calix[6]arene to disturb the release of EVs and the activity of MMPs in pancreatic cancer cells. We found a correlation between the endocytic-associated mediators and the prognosis of pancreatic cancer patients. We observed a more active EV machinery in the pancreatic cancer cell line PANC-1, which was reduced three-fold by treatment with calix[6]arene at subtoxic concentration (5 μM; p 〈0,001). We observed the modulation of 186 microRNAs (164 miRNAs upregulated and 22 miRNAs downregulated) upon calix[6]arene treatment. Interestingly, some of them as miR-4443 and miR-3909, regulates genes HIF1A e KIF13A that are well known to play a role in transport of vesicles. Furthermore, Calix[6]arene downmodulated matrix metalloproteinases (MMPs) -2 and − 9 and disturbed the viability of pancreatic organoids which recapitulate the cellular heterogeneity, structure, and functions of primary tissues. Our findings shed new insights on calix[6]arene's antitumor mechanism, including its intracellular effects on vesicle production and trafficking, as well as MMP activity, which may harm the tumor microenvironment and contribute to a reduction in cancer cell dissemination, which is one of the challenges associated with high mortality in pancreatic cancer.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

Abstract LB158: Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy

Abstract Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines and comparison of the effects of each drug combination with the effect of the individual drugs are shown in Table. The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes. Table. Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines. Comparison of the effects of each drug combination with the effect of the individual drugs (Table shows the p-values for each comparison.) Colony formation BxPC-3 PL45 Capan-1 Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano+TLZ 0.00048 0.0052 2.40E-06 2.70E-06 0.0051 0.021 Pano+Ola 0.0035 0.078 0.0011 0.00094 0.0016 0.022 SAHA+TLZ 1.80E-05 0.17 0.0029 0.012 0.0014 0.019 SAHA+Ola 0.00018 0.014 0.0016 0.0068 0.0066 0.029 Pano+TLZ+DAC 4.20E-05 0.00026 0.0015 1.70E-05 1.20E-05 2.70E-06 0.00052 0.00088 0.0064 Pano+Ola+DAC 3.40E-05 0.00049 0.0013 7.10E-07 1.70E-06 2.20E-06 0.00068 0.0028 0.0053 SAHA+TLZ+DAC 0.00016 0.00016 0.0011 1.90E-05 3.70E-06 9.30E-07 0.00018 0.00017 0.011 SAHA+Ola+DAC 4.90E-07 0.00035 0.001 0.00022 0.00026 0.00084 0.00016 0.0016 0.0035 Drug-mediated inhibition of cell proliferation Pano+TLZ+DAC 3.57E-05 3.89E-07 1.30E-07 2.91E-05 3.76E-06 1.80E-08 0.0239 ND 0.0092 Pano+Ola+DAC 7.76E-05 1.43E-07 2.52E-07 6.59E-07 1.03E-08 2.22E-07 0.0013 0.21 0.0034 SAHA+TLZ+DAC 3.04E-06 0.00034 1.17E-05 4.91E-08 4.45E-06 1.74E-07 0.0013 1.75E-01 0.007 SAHA+Ola+DAC 0.00012 0.0032 0.000068 9.64E-09 1.53E-08 9.35E-07 0.0007 0.18 0.0054 Abbreviations: DAC: decitabine; ND: not determined; Ola: olaparib; Pano: panobinostat; SAHA: vorinostat; TLZ: talazoparib P ≤ 0.05 is considered statistically significant. Citation Format: Apostolia M. Tsimberidou, Benigno C. Valdez, Bin Yuan, Yago Nieto, Mehmet Baysal, Abhijit Chakraborty, Borje S. Andersson. Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB158.

Abstract LB397: Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation

Abstract CRISPR-Cas9, a highly effective gene editing tool, is a molecular scissor that induces double strand breaks (DSBs) at its targeted genomic locations. Ionizing radiation (IR), in contrast, generates DSBs and other DNA lesions, making it potentially more toxic than CRISPR-Cas9 induced DSBs. Alternatively, CRISPR-Cas9 might be more toxic as the Cas9 activity would continue if the DSB were repaired to wild type, resulting in more scissions per target. We designed multitarget sgRNAs targeting 0-16 sites in the human genome and performed similar dose-response experiments with IR (0-10 Gy) over 21 days on two pancreatic cancer (PC) cell lines. Growth inhibition was measured through clonogenicity and alamarBlue assays, and γ-H2AX staining performed to calculate DSBs. Surviving colonies from sgRNA transduction underwent whole genome sequencing (WGS) and targeted deep sequencing to quantify mutations. Cell growth was assessed through cell counting and sgRNA tag survival over time. Chromosomal changes were elucidated through chromosome breakage assays and XY FISH. We found that cytotoxicity increased as a function of both IR dose and number of sgRNA target site. At a comparable number of DSBs, CRISPR-Cas9 demonstrated higher cytotoxicity (~44% growth inhibition from 5-target sgRNA, ~15 γ-H2AX foci in the PC cells) compared to IR (9% inhibition from 0.5 Gy, ~25 foci). WGS of 5-cutter treated cells also revealed ~13 CRISPR-Cas9 induced DSBs in the PC cells. >99% cytotoxicity was achieved with 12-target sgRNA and 8 Gy. Moreover, sequencing of surviving colonies from sgRNA-treated cells confirmed lack of widespread off-target events. Notably, no surviving colonies could be obtained from the 12- and 16-cutter treated Panc10.05 cells and 8-14 cutters treated TS0111 cells.Both CRISPR-Cas9 and IR assays demonstrated delayed cell death, and cell death began 7 days after transduction of multitarget sgRNAs. Cytogenetic analyses on cells transduced with a 14-cutter sgRNA over 21 days revealed accumulation of karyotypic abnormalities peaking at day 14, and polyploidization peaking at day 10 and decreasing by day 21. Structural variant (SV) analyses from WGS data showed that the number of SVs per colony increased and peaked in 6-cutter colonies (average = 42 SVs), then decreased in 8- and 14-cutter colonies. Finally, surviving colonies that were resistant to 5-, 8-, and 16-cutter sgRNAs were re-challenged with their original multitarget sgRNA, 12-, and 14-cutter sgRNAs. Re-treating with original sgRNA showed no growth inhibition, while >95% cytotoxicity was observed by re-challenging them with 12- or 14-cutter sgRNAs.In conclusion, CRISPR-Cas9 kills pancreatic cancers by inducing chromosomal catastrophe, and resistance to original set of CRISPR-Cas9 induced DSBs can be easily overcome by targeting different sites. For the same number of DSBs, CRISPR-Cas9 was surprisingly more toxic than IR. Citation Format: Selina Shiqing K. Teh, Akhil Kotwal, Eitan Halper-Stromberg, Laura Morsberger, Kirsten Bowland, Alexis Bennett, Michael Goldstein, Ying S. Zou, James R. Eshleman. Simultaneous CRISPR-Cas9 induced double strand breaks causes chromosomal catastrophe in pancreatic cancers: A parallel comparison with ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB397.

Abstract LB203: CRISPR anchor screens to identify co-vulnerabilities and drug combinations in DepMap

Abstract The Cancer Dependency Map (DepMap) aims to systematically map the landscape of cancer vulnerabilities by profiling a wide range of cancer cell lines that collectively represent the heterogeneity of human cancer. An increasing number of targets and biomarkers emerging from this effort are in drug development or clinical trials. However, it is clear that drug resistance is a significant challenge and that drug combinations will be required. CRISPR anchor screens involve introducing an "anchor" which can be either a drug or genetic perturbation, alongside the sgRNA library. This approach provides a way to investigate resistance mechanisms, potential drug combinations, or co-vulnerabilities. As a proof of concept, here we describe CRISPR anchor screens with MRTX1133 as the anchor, which is a potent and selective KRASG12D inhibitor currently in clinical trials. We performed genome-scale loss-of-function CRISPR-Cas12a screens in the presence of MRTX1133 in pancreatic and lung cancer cell lines that harbor the KRASG12D. The goal of these screens was to identify cancer dependencies that are either present or enhanced in the presence of MRTX1133. We find multiple such MRTX1133-induced dependencies that are either common across all cell lines screened or unique to one or a few. This shows that the response to MRTX1133 can vary depending on the context, highlighting the importance of finding biomarkers to predict the response. Identifying the biomarkers of these context-specific dependencies would provide strategies to select patient populations that would respond to the drug combinations. This study not only provides valuable insights into enhancing the efficacy of KRAS-targeted therapies but also serves as a pioneering effort in incorporating innovative CRISPR screen modalities into the DepMap. Citation Format: Jenny Serra-Vinardell, Zoe Posner, Alison Cameron, Yuqi Chen, Negin Karisani, Barbara De Kegel, Joshua M. Dempster, Andrew J. Aguirre, Francisca Vazquez. CRISPR anchor screens to identify co-vulnerabilities and drug combinations in DepMap [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB203.

Abstract LB192: AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid malignancy with low survival rates due to a dense stroma, abnormal gene expression patterns, and limited treatment options. Anterior gradient 2 (AGR2) is often overexpressed in many cancers, including PDAC. AGR2 belongs to the protein disulfide isomerase family of proteins and is located in the endoplasmic reticulum, where it helps with protein-disulfide isomerase reactions to ensure proper protein folding. However, the molecular mechanisms underpinning AGR2 overexpression in PDAC pathophysiology are not well understood. Gene expression data obtained from the pancreatic adenocarcinoma (PAAD) dataset shows that AGR2 is overexpressed in PDAC tumors compared to normal tissues. We confirmed this gene over-expression in PDAC tumors and multiple pancreatic cancer cell lines using fluorescent microscopy, RT-qPCR, and western blotting. Using stable inducible short hairpin (shRNA) technology, we showed that AGR2 knockdown significantly decreases the viability and colony-forming ability of HPAF-II and PANC-1 cells. Gene Ontology (GO) enrichment analysis revealed that AGR2 knockdown suppresses a host of protein biosynthesis pathways, including protein targeting and localization to the endoplasmic reticulum (ER). Significantly, ER stress was induced in knockdown cells, shown by the activation of unfolded protein response, suggesting an accumulation of unfolded proteins. To determine the effects of unfolded protein accumulation on the ER, we performed transmission electron microscopy. Here, the results show an increase in the ER lumen size of knockdown cells compared to controls. Interestingly, we observed a decrease in mitochondria size and confirmed the increased expression of mitochondria fission proteins DRP1 and MFF. Finally, flow cytometry analysis revealed increased apoptosis in AGR2 knockdown cells compared to controls. Overall, our data provide insights into the role of AGR2 in PDAC tumorigenesis. However, more research is warranted to determine if AGR2 could be a potential therapeutic target. Citation Format: Philip Salu, Katie Reindl. AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB192.

Abstract LB274: Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression

Abstract HIKESHI, under heat stress conditions, has been reported to transport molecular chaperone HSP70 into the nucleus to prevent cancer cell death from heat stress. The tumor-supporting roles of molecular chaperones in various solid tumors are explored extensively. Yet, the sole targeting of the molecular chaperones could not significantly impact anticancer treatment. Thus, we hypothesized that the specific transporter protein HIKESHI might improve this outcome. However, understanding of the clinical relevance of HIKESHI in pancreatic cancer remained unclear, and few studies addressed the HIKESHI role in cancer and whether targeting can inhibit pancreatic cancer cell viability and the other HSPs. Here, we first investigated the expression pattern of the HIKESHI protein in surgically resected pancreatic cancer samples (n=142) and analyzed the clinical relevance of the HIKESHI. We found that the HIKESHI protein was overexpressed in cancer compared to the adjacent normal tissue, and positive expression of cancer HIKESHI was related to poor overall survival. In addition, HIKESHI-positive expression in cancer was identified as an independent prognostic marker for overall survival. Next, using shRNA, we established the stable HIKESHI-suppressed pancreatic cancer cell line (PANC-1). HIKESHI suppression inhibited cell growth and colony formation ability in vitro. The expression and nuclear translocation of HSP70 and HSP110/105 were suppressed in HIKESHI-suppressed cells compared to the control shRNA cells despite no heat stress conditions. Furthermore, we found that the tumor growth, HIKESHI, and Ki67 expression in HIKESHI-suppressed tumors were inhibited compared to the control shRNA tumors. Next, we performed Cap Analysis of Gene Expression and IP-MS analysis to discover the potential downstream and novel interactive partner of the HIKESHI protein. The extensive mechanism of these analyses is under investigation. Our findings suggest that targeting HIKESHI may be a promising therapeutic strategy against refractory pancreatic cancer. Citation Format: Bilguun Erkhem-Ochir, Takehiko Yokobori, Gendensuren Dorjkhorloo, Haruka Okami, Takaomi Seki, Norifumi Harimoto, Ryo Muranushi, Kouki Hoshino, Kei Hagiwara, Norihiro Ishii, Mariko Tsukagoshi, Kenichiro Araki, Hiroshi Saeki, Ken Shirabe. Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB274.

Senolysis of gemcitabine-induced senescent human pancreatic cancer cells.

Gemcitabine (GEM) is often used to treat pancreatic cancer. Many anti-cancer drugs induce cancer cell death, but some cells survive after cell cycle arrest. Such a response to DNA damage is termed cellular senescence. Certain drugs, including the Bcl-2-family inhibitor ABT-263, kill senescent cells; this is termed senolysis. In this study, we examined the therapeutic benefits of ABT-263 in GEM-induced senescence of human pancreatic cancer cells. Of four pancreatic cancer cell lines (PANC-1, AsPC-1, CFPAC-1, and PANC10.05), GEM induced senescent features in PANC-1 and AsPC-1 cells, including increases in the cell sizes and expression levels of mRNAs encoding interleukin (IL)-6/IL-8 and induction of β-galactosidase. Successive treatment with GEM and ABT-263 triggered apoptosis in PANC-1 and AsPC-1 cells and suppressed colony formation significantly. Senolysis of GEM-induced senescent pancreatic cancer cells by ABT-263 was triggered by a Bcl-xL inhibitor, but not by a Bcl-2 inhibitor, suggesting a central role for Bcl-xL in senolysis. In a xenograft mouse model, combined treatment with GEM and ABT-737 (an ABT-263 analog exhibiting the same specificity) suppressed in vivo growth of AsPC-1 significantly. Together, our results indicate that sequential treatment with GEM and senolytic drugs effectively kill human pancreatic cancer cells.

MSLN induced EMT, cancer stem cell traits and chemotherapy resistance of pancreatic cancer cells

Chemoresistance is one of the main reasons for poor prognosis of pancreatic cancer. The effects of mesothelin (MSLN) on chemoresistance in pancreatic cancer are still unclear. We aim to investigate potential roles of MSLN in chemoresistance and its relationship with proliferation, epithelial-mesenchymal transition (EMT) and cancer stemness of pancreatic cancer cells. Human pancreatic cancer cell lines ASPC-1 and Mia PaCa-2 with high and low expression of MSLN, respectively, were selected. The ASPC-1 with MSLN knockout (KO) and Mia PaCa-2 of MSLN overexpression (OE) were generated. The effects of MSLN on cell phenotypes, expression of EMT-related markers, clone formation, tumor sphere formation, and pathologic role of MSLN in tumorigenesis were detected. Sensitivity of tumor cells to gemcitabine was evaluated. The results showed that adhesion, proliferation, migration and invasion were decreased significantly in ASPC-1 with MSLN KO, whereas increased significantly in Mia PaCa-2 with MSLN OE. The size and the number of clones and tumor spheres were decreased in ASPC-1 with MSLN KO, and increased in Mia PaCa-2 with MSLN OE. In xenograft model, tumor volume was decreased (tumor grew slower) in MSLN KO group compared to control group, while increased in MSLN OE group. Mia PaCa-2 with MSLN OE had a higher IC50 of gemcitabine, while ASPC-1 with MSLN KO had a lower IC50. We concluded that MSLN could induce chemoresistance by enhancing migration, invasion, EMT and cancer stem cell traits of pancreatic cancer cells. Targeting MSLN could represent a promising therapeutic strategy for reversing EMT and chemoresistance in pancreatic cancer cells.

The DDX6/KIFC1 signaling axis, as regulated by YY1, contributes to the malignant behavior of pancreatic cancer.

Human DEAD/H box RNA helicase DDX6 acts as an oncogene in several different types of cancer, where it participates in RNA processing. Nevertheless, the role of DDX6 in pancreatic cancer (PC), together with the underlying mechanism, has yet to be fully elucidated. In the present study, compared with adjacent tissues, the level of DDX6 was abnormally increased in human PC tissues, and this increased level of expression was associated with poor prognosis. Furthermore, the role of DDX6 in PC was investigated by overexpressing or silencing the DDX6 in the PC cell lines, SW1990 and PaTu-8988t. A xenograft model was established by injecting nude mice with either DDX6-overexpressing or DDX6-silenced SW1990 cells. DDX6 overexpression promoted the proliferation and cell cycle transition, inhibited the cell apoptosis of PC cells, and accelerated tumor formation, whereas DDX6 knockdown elicited the opposite effects. DDX6 exerted positive effects on PC. RNA immunoprecipitation assay showed that DDX6 bound to kinesin family member C1 (KIFC1) mRNA, which was further confirmed by RNA pull-down assay. These results suggested that DDX6 positively regulated the expression of KIFC1. KIFC1 overexpression enhanced the proliferative capability of PC cells with DDX6 knockdown and inhibited their apoptosis. By contrast, DDX6 overexpression reversed the inhibitory effect of KIFC1 silencing on tumor proliferation. Subsequently, the transcription factor Yin Yang 1 (YY1) was shown to negatively regulate DDX6 at both the mRNA and protein levels. Dual-luciferase reporter assay verified that YY1 targeted the promoter of DDX6 and inhibited its transcription. High expression levels of YY1 decreased the proliferation of PC cells and promoted cell apoptosis, although these effects were reversed by DDX6 overexpression. Taken together, YY1 may target the DDX6/KIFC1 axis, thereby negatively regulating its expression, leading to an inhibitory effect on pancreatic tumor.

Synchrotron radiation-FTIR biomolecular assessment in PANC-1 and MIA PaCa-2 pancreatic cancer cells after treatment with Gd-TiO2 nanoparticles

The increasing incidence rate of pancreatic cancer worldwide, along with high resistance to existing chemotherapies and radiotherapy, demand new approaches for its treatment. Nanoparticle-guided cancer treatment modalities are promising in their anti-cancer efficiency and upgrading potential. We have synthesized and characterized Gd-doped titanium-dioxide nanoparticles (Gd-TiO2 NPs), which showed good biocompatibility with MIA PaCa-2 and PANC-1 pancreatic cancer cell lines. To assess the effect that these NPs have on tumor cells, we used synchrotron radiation Fourier transform infrared spectroscopy and principal component analysis. We elucidated the biochemical changes in the two cell lines induced after their interaction with these NPs. Results demonstrated changes in nucleic acids and carbohydrate metabolism and alterations in the protein conformation in both cell lines. In addition, considerable changes in lipid area were observed in PANC-1 cell line, suggesting that Gd-TiO2 NPs have a prospective to be further studied as systems for pancreatic cancer treatment.

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

BackgroundPreviously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. MethodsWe engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/β-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. ResultsHyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating β-catenin activation. Overexpression of EFNA4 activated the β-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity—a phenomenon remediated by the β-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/β-catenin/dCK axis. ConclusionThis study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/β-catenin axis and activation of dCK.